Classical swine fever (CSF) is an epizootic animal disease that occurs worldwide and has significant political and economic relevance (Vandeputte and Chappuis, 1999). Classical Swine fever, also known as hog cholera or pig plague, is one of the diseases which must be notified Nationally, on the EU-level as well as to the World Organization for Animal Health (OIE) in Paris upon appearance in any member state. Classical swine fever is caused by a small enveloped RNA-virus of the genus Pestivirus in the family Flaviviridae. The natural hosts of the swine fever virus are solely domesticated and wild swine species (e.g. European wild boar).
Attempts have been made within the European Union to eradicate CSF through rigorous measures without prophylactic vaccination, which has been forbidden since 1990. Despite the prohibition, vaccination does represent a legally approved option as an emergency vaccination in cases when swine fever appears. In such an event the vaccination should occur via one of the emergency vaccination plans, which have been ratified by the European Union (see Art. 19 of the Counsel Directive 2001/89/EC). Up until now, Romania has been the only country in which an emergency vaccination has been carried out. The reasons for such limited application lie with technical limitations of the marker vaccines available at the present time, such as restrictions in vaccine efficacy, in addition to trade barriers relating to conventionally vaccinated animals (limitation to national marketing). The efficiency of the licensed marker vaccines cannot be compared with modified live vaccines, which exhibit significant advantages, and such inactivated vaccines or subunit vaccines are anyway not suited for emergency vaccination due to later onset of immunity and the need of re-vaccinations.
Considering the expansion of the European Union towards countries in Eastern Europe and ever-increasing globalisation, new strategies have been discussed for potential emergency vaccination, which will play a role in avoiding large scale culling of animals and associated economic losses (Leifer et al., 2009). There is therefore a significant demand for a highly efficient vaccine which allows serological differentiation between vaccinated and non-vaccinated animals and furthermore exhibit all the advantages of traditional modified live vaccines.
Because the first generation of marker vaccines, which were based on the E2 glycoprotein of the CSF virus have only a restricted availability and severe disadvantages like storage conditions, costs, efficacy, there is a large demand for novel marker vaccines. Various candidates for such vaccines have been investigated, such as DNA vaccines, immunogenic peptides, vector vaccines, deletion mutants and chimeric fever viruses (Beer et al., 2007; Dong and Chen, 2007). Most of these marker vaccine candidates exhibit the disadvantage that they are produced via modern methods of genetic modification. Due to the significant consumer fear of genetically modified products, in addition to complicated admission procedures, genetically modified vaccines exhibit significant disadvantages.
Traditional vaccines directed against CSFV do include modified live vaccines. Such vaccines are highly efficient after single application but do not allow the differentiation between vaccinated and infected animals on the basis of a serological profile. Many of these vaccines are based on the classic viral strain “C” or a derivative thereof (so-called “C-strain vaccines”). Additionally, there exist vaccines based on Japanese viral strain “guinea pig exultation-negative (GPE−)”, the “Thiverval” strain and the “Mexican PAV” strain, all of which have been used in both regional and international settings (Biome et al, 2006; Greiser-Wilke & Moennig, 2004; van Oirschot, 2003). Extensive data do exist regarding use of the C-strain-based vaccines. It is known that four days after application of the vaccine, a complete protection of the animals against virulent CSFV challenge infection can be demonstrated. Additionally, seven days after vaccination, a complete protection is provided from vertical transmission of challenge virus in carrier animals (de Smit et al., 2001).
The significant disadvantage of the known modified living vaccines is the absolute inability to serologically discriminate between vaccinated and infected animals. In light of this, one task of the present invention is to provide a modified living vaccine which enables discrimination between vaccinated and infected animals.
In the area of marker vaccines, the so called sub-unit vaccines are known in the prior art, which are based upon the recombinant E2 glycoprotein of CSFV. The discrimination test for such vaccines is the enzyme-linked immunosorbent assay (ELISA), in which antibodies directed against the Ems glycoprotein are used to indirectly detect CSF virus infections. At the present time, only one E2-subunit vaccine is available on the market, however, the license was suspended for some months (see EMA report on E2 subunit vaccines).
Regardless of the inability to market such products, such systems exhibit grave biological disadvantages. One such disadvantage is that at least two parenteral immunisations are required before complete protection is conveyed, which renders such vaccines completely incompatible with “bait-vaccinations”, where animals are fed in a single dose with vaccine baits. In addition, emergency vaccination campaigns are only reasonable when a protection against wild type CSFV In various experiments using the E2 subunit vaccines, complete protection from vertical transmission was not achieved. A further disadvantage of such systems is that antibodies directed against the glycoprotein Ems are used for serological differentiation and the sensitivity and specificity of such test systems was only ever moderate (Floegel-Niesmann, 2003).
Live attenuated CSF vaccines are known in the art but have until now been hindered by various disadvantages. Most vaccines disclosed in the prior art comprise of foreign DNA and are generated using methods of genetic engineering, otherwise known as recombinant DNA technology, therefore associating serious environmental risk assessment problems with the product. Other CSFV variants are known where amino acids are either substituted or deleted from the wild-type TAV epitope (WO 2010/074575 A2). However, the amino acid residues and/or nucleotides that are modified in the present invention are neither disclosed nor suggested in the prior art.
Multiple passaging has also been used to generate virus variants, which may be used as vaccines, although antibody pressure has not been previously applied. The multiple passaging of virus-infected cultures in order to generate variants as disclosed in the prior art is therefore limited by having to conduct a large number of culture passages and also by a lack of control over which epitope is to be modified (Hulst et al).
Kortekaas et al describe a genetically stable, live attenuated CSF vaccine, which enables the serological differentiation of infected from vaccinated animals. A mutated C-strain was genetically modified using a targeted approach, whereby recombinant DNA technology was used to introduce deletions into the E2 protein of the CSFV. Further mutations were subsequently acquired at various locations within the virus genome via multiple passaging to create strains exhibiting enhanced proliferation. The strains disclosed in Kortekaas et at are genetically engineered viruses, which is a significant disadvantage in light of the complicated admission processes for releasing genetically modified products into the environment.
Holinka et al disclose a double antigenic marker live attenuated CSFV strain “FlagT4vn” which was obtained by combining two genetic determinants of attenuation. FlagT4v harbors a positive antigenic marker, synthetic Flag epitope, introduced via a 19mer insertion in E1 glycoprotein; and a negative marker resulting from mutations of the binding site of monoclonal antibody WH303 (mAbWH303) epitope in the E2 glycoprotein. Intranasal or intramuscular administration of FlagT4v protected swine against virulent CSFV Brescia strain at early (2 or 3 days), and late (28 days) time postinoculation. FlagT4v induced antibody response in pigs reacted strongly against the Flag epitope but failed to inhibit binding of mAbWH303 to a synthetic peptide representing the WH303 epitope. The vaccine disclosed in Holinka relates to a genetically engineered virus that exhibits foreign DNA within its genome (Flag-Tag sequence in addition to associated vector sequence and markers). This represents a significant disadvantage compared to the present invention, which exhibits no foreign or recombinant DNA.
The document WO 2007/143442 A2 describes the effects of mutations within the WH303 epitope of CSFV E2, which change the amino acid sequence of the virulent Brescia CSFV progressively toward the homologous amino acid sequence of BVDV strain NADL. Animals infected with virus mutants were protected when challenged with virulent Brescia virus at 3 and 21 days post vaccination. Modification at this site within the WH303 epitope also allows development of a diagnostic test to differentiate vaccinated from infected animals. Despite these effects, the mutations were introduced using genetic engineering, therefore introducing foreign genetic material into the viral vaccine.
The aforementioned state of the art discloses vaccines that have been engineered via recombinant genetic technology to produce the virus strains. As described above, genetically engineered vaccines are subject to environmental safety concerns and are therefore hindered by complicated admission protocols and fear amongst the general public, therefore providing significant disadvantage to their use.